Ethyl Acetate Fraction of Caesalpinia sappan L. Enhances Cisplatin s Cytotoxicity on HeLa Cells via G1 and S Arrest through p53 Expression

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1 Indonesian Journal of Cancer Chemoprevention, 2017, 8(2): ISSN: Ethyl Acetate Fraction of Caesalpinia sappan L. Enhances Cisplatin s Cytotoxicity on HeLa Cells via G1 and S Arrest through p53 Expression Ulfatul Husnaa 1,2, Ni Putu Linda Laksmiani 2,3, Ratna Asmah Susidarti 2,4, Edy Meiyanto 2,4 1 Master Course Program of Biotechnology, Graduate School, Universitas Gadjah Mada, Yogyakarta 2 Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 3 Department of Pharmacy, Faculty of Mathematics and Science, Universitas Udayana, Bali 4 Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta Abstract Cisplatin (cisp) is the first line chemotherapeutic agent for several cancer diseases which can cause significant side effects and cellular resistance. Combination-chemotherapy treatment (co-chemotherapy) was reported to be able to reduce cisp effects. Therefore, this study was carried out to investigate the cytotoxic activity of ethyl acetate fraction of C. sappan (EFC) in combination with cisp by observing apoptosis induction and cell cycle profile. Cytotoxic activity was evaluated by MTT assay. Cell cycle and apoptosis analysis were performed using flow cytometry and p53 expression was analyzed using immunocytochemistry. EFC performed cytotoxic effect on HeLa cells by showing morphological changes such as cell shrinkage, rounding and decreasing of cells viability in concentration dependent manner, giving IC50 value of 65 μg/ml. Combination of EFC and cisp in low concentration decreased cell viability into 36.86%. Further assay indicated that this combination caused redistribution of cell cycle arrest in G1 and S phases through p53 stabilization in nucleus. However, that mechanism was not followed by apoptosis. These results provide evidence to support EFC development as the enhancer of cisp effect, by improving its cytotoxicity on HeLa cells. EFC increases HeLa cells sensitivity to cisp through G1 and S cells arrest depending on p53 expression. Key words: co-chemotherapy, EFC, cervix cancer HeLa cells, p53, G1 and S arrest. INTRODUCTION Cervical cancer is the third most common disease among women worldwide (Jemal et al., 2011), which 70% of its occurrence is caused by HPV infection (Castellsagué, 2008). Globally, 80-85% of death occurs in low- and middle-economicincome countries. The statistics estimation was made based on Globocan 2008 which state that the incidence of cervical cancer is increasing every year, with global estimation of new cases expected to increase 2% per annum to 770,000 new cases by 2030 (Forman et al., 2012). There are several ways to treat cervical cancer, including surgery, radiation therapy, chemotherapy, and combination of those treatments. To date, chemotherapeutic agent is the most common treatment (Martin et al., 2013; Leaver and Labonte, 2010). Cisplatin (cisp) is the first chemotherapeutic agent from platinum (Jamieson and Lippard, 1999). This agent caused many side effects, such as severe nausea and vomiting, acute and chronic nephrotoxicity, anemia (Miller et al., 2010; Rabik and Dolan, 2007), acute cochlear toxicity (ototoxicity) (Mukherjea and Rybak, 2011) and also developed cells resistance (Sirichanchuen et al., 2012). Some of natural compounds were known as cytotoxic agent and can be combined with chemotherapeutic agent. Therefore, cisp s side effects and cellular resistance caused by cisplatin can be reduced using chemotherapeutic combination with natural agent by decreasing of cisplatin uses (Zhang et al., 2011; Zhao et al., 2004). Corresponding author meiyan_e@ugm.ac.id 51

2 Herbal medicines used in nutrient supplements as anti-cancer and antiinflammatory agents give benefit for patients (Ho et al., 2002) to overcome side effects and drug resistance (Zhang et al., 2011). The heartwood of Caesalpinia sappan has been used traditionally for medical treatment in Asia, consumed as a drink or a dye (Badami et al., 2004). Previous studies showed that C. sappan extract (Ueda et al., 2002) and its isolated compounds, brazilin and brazilein, potentially exhibited antitumor activity on several rodents and tumor cancer cells (Ren et al., 2011; Yen et al., 2011), but not on normal epithelial cells (Lee et al., 2010). The main constituent of C. sappan, brazilin, can be oxidized by air and light to produce brazilein that is soluble in ethyl acetate (Kim et al., 1997). Therefore, in this study, the effects of EFC-single treatment and in combination with cisp on HeLa cells were determined. Observation was done by observing cell viability, apoptosis induction, and cell cycle progression. Expression of p53 protein level was also investigated. More research in field of science will help for further explore the effectiveness and safety approaches, as both agents in conventional treatment and herbal medicine. METHODS Preparation of EFC Caesalpinia sappan heartwood powder was obtained from Balai Besar Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional (B2P2TOOT), Tawangmangu, Indonesia. C. Sappan wood was extracted with methanol, evaporated, and partitioned with n-hexane and ethyl acetate by liquidliquid extraction. Ethyl acetate fraction was evaporated by using rotary evaporator to get the residue as EFC. The dried fraction was resolubilized in dimethyl sulfoxide (DMSO) before used as treatment sample. Cisplatin (Wako, 98% HPLC, Japan, CAS No ) was used as chemotherapeutic agent. Chemicals and reagents DMEM (Dulbecco s Modified Eagle Media, low glucose), FBS (Fetal Bovine Serum), penicillin/streptomycin, fungizone were purchased from Gibco (Invitrogen, USA). MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide), PI (Propidium Iodide) (minimum 95% (HPLC) were purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA). DMSO (99.5% pro GC, Sigma Aldrich Chemie GmBH, Steinheim, Germany), trypsin-edta (Gibco, Invitrogen, Canada), FITC-Annexin V Apoptosis Detection Kit (Biovision, USA), Triton-X (triton X-100 for GC, E.Merck, 64271, Darmstadt, Germany). SDS (Sodium Dodecyl Sulphate), HCl were purchased from Merck, Germany. RNase was obtained from Lab. Animal Sciences, NAIST, Japan. All other chemicals were used under analytical grade. Cell culture HeLa cells were obtained from Prof. Masashi Kawaichi, Laboratorium of Gene Function in Animal, Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), Japan. Cells were maintained in DMEM-low glucose, supplemented with 10% (v/v) FBS, 1.5% (v/v) penicillin-streptomycin, and 0.5 % (v/v) fungizone. Cells were incubated at 37 C and 5% CO 2. MTT assay HeLa Cells were grown 12x10 3 onto 96-well plates before being exposured to drug treatment. For cell viability assay, cells were treated for 24 h with increasing concentration of EFC-single treatment (0, 30, 40, 50, 60, 70, 80, 90 μg/ml). Combination treatment was done using both of EFC and cisp, incubated for 24 h. As negative control, the cells were only treated with growth medium. At 100 µl media containing MTT (0.5 mg/ml) was added to each well then incubated for 3 h at 37 C. The reaction was stopped by dilution with 10% (w/v) SDS in 0.01 M HCl, then continued by incubation for overnight. The absorbance was determined by using ELISA reader (Bio-Rad microplate reader Benchmark, serial no , Japan) at λ 595 nm as previously described with some modification (Mosmann, 1983). Flow cytometry FACS analysis was carried out to investigate apoptosis quantification and cell cycle distribution. Around 5 x 10 5 cells/well were grown in 6-well plates and cells were treated with EFC, cisp, and combination of both compounds for 24 h. Trypsinized 52

3 adherent cells were collected and were prepared for detection. Cell apoptosis was detected by using a FITC-Annexin V Apoptosis Detection Kit (Biovision, USA). Then, apoptosis was analyzed by flow cytometry (BD FACS-Calibur, USA). For cell cycle analysis, the trypsinized adherent cells were collected and detected by adding flow cytometry reagents (25µl PI, 2.5µl RNase, 0.5µl Triton-X in 500µl PBS), followed by incubation at room temperature for 10 minutes in the dark. The cell suspension was transferred into a flow cytometer tube and its cell cycle profile was read using flow cytometry. The cell cycle profile was analyzed by ModFit LT 3.0. Immunocytochemistry Immunocytochemistry was developed (Cozmei et al., 2002) with some modifications. HeLa cells were grown with the density of 5 x 10 4 cells/cover slip in 24- well plate for 24 h prior to treatment of EFC, cisp, and combination of both. Cells were treated for 15 h and then fixed with cold methanol for 10 minutes and washed twice using PBS and sterile water. Cells were blocked with H 2O 2 and then pre-diluted blocking serum was added, continued with incubation for 10 minutes. Cells were washed using PBS and incubated with primary monoclonal antibody anti-p53 (mouse monoclonal anti-p53 antibody, BioGenex) overnight. Cells were washed using PBS, then universal detection kit was added (Star Trek Universal HRP Detection Kit, Ref STUHRP 700L10-KIT, Biocare Medical). Cells were then incubated with biotinylated universal secondary antibody for 20 minutes, followed by streptavidin-enzyme horse radish peroxidase for 10 minutes. Substrate solution chromogen 3,3 -diaminobenzidine (DAB) was exposed for 10 minutes and cells were counterstained with Mayer s Haematoxylin (Dako) for 1 minute. Then, cells were fixed with ethanol and xylol. Between each immunostaining step, cells were washed briefly in PBS ph 7.4. Negative controls were prepared by replacing the primary antibody with PBS. The p53 expression on the cells was observed by binocular microscope (Nikon YS 100, Japan). Statistical analysis Single and combinational cytotoxicity assay: IC 50 value was calculated by equation y=bx+a acquired from linear regression between concentration and % cell viability. IC 50 is concentration inhibiting 50% cell proliferation. Cell viability was calculated with the formula (Meiyanto et al., 2011): % Cells viability = Treated cells abs medium control abs x 100% Cells control abs medium control abs The datum were performed as average ±SE from triplicate (n = 3) experiments. Cell viability and p53 expression were statistically analyzed using one way ANOVA and statistical significance was estimated by using Tukey HSD. Statistical significance was determined at P < RESULT Cytotoxic EFC and Its Combination with Cisp on HeLa cells Cytotoxicity assay was used to evaluate the potential of EFC cytotoxicity on HeLa cells. Furthermore, IC 50 value was acquired as the parameter of EFC concentration to inhibit 50% HeLa cells growth. Figure 1 shows the effect of EFC to cell viability for 24 h treatment. EFC caused inhibition on cell growth in a concentrationdependent manner (Figure 1A). We suggest that EFC was found to be cytotoxic on HeLa cells with IC 50 value of 65±1.5 µg/ml. This fraction exhibited potent cytotoxicity (IC µg/ml) (Prayong et al., 2008). Treated cells showed cytotoxic effect with changes in cell morphology and the decrease of cell viability. Viable cells exhibited epithelial shape, but after being treated with certain concentration of samples, the cells exhibited cell shrinkage and rounding (Figure 1B). Cisp performed cytotoxic activity on HeLa cells with IC 50 value of 16 µm (data not showed). IC 50 value was then used to determine the concentration in combinational treatment that is concentration lower than IC 50 value. Various concentrations of cisp (2, 4, 8 µm) were combined with 28 µg/ml EFC. Figure 2 sections confirm that combinational treatment performed morphological changes and caused decrease of the cells number. Cell viability measurement of the combination group (EFC combined to cisp) was 53

4 considered significantly different compared to cisp-single treatment group (P < 0.05). At 8 µm cisp in combination with 28 µg/ml EFC decreased significantly HeLa cells viability compared to 2 and 4 µm cisp (P < 0.05). There was approximately 63% decrease in HeLa cells viability treated by 28 µg/ml EFC combined 8 µm cisp for 24 h. Cell viability acquired from single treatment of EFC and cisp above 100% was considered as 100%. Therefore, both low-concentration of EFC and cisp showed no inhibitory effect on HeLa cells. Whereas, combination between EFC and cisp caused a significant decrease in cell viability. This result indicates that EFC can be used in combination with cisp on HeLa cells. (A) (B) control EFC (μg/ml) Figure 1. Effects of EFC on cell viability and morphological changes on HeLa cells. (A) HeLa cells was treated using EFC (0,30-90 μg/ml) for 24 h and determined by MTT assay as described in materials and methods. Data are representative of values from three independent experiments. (B) Morphological changes of HeLa cells were observed using binocular microscope. Viable cells (full arrow), morphological changes (dotted line), bars: 50 µm (magnification 100x) 54

5 (A) cell viability 93% 72% 37% Cisp 2 µm Cisp 4 µm Cisp 8 µm EFC 28 µg/ml (B) control 4 μm cisp 2 μm cisp 8 μm cisp 28 μg/ml EFC Figure 2. Cytotoxic Effect of EFC was combined with cisp on HeLa cells. (A) HeLa cells were treated for 24 h with combination both of EFC and cisp caused decreasing percentage of cell viability. Cells viability were determined by MTT assay as described in materials and methods. () Significances (P < 0.05). (B) Cells morphological changes treated with combination both of EFC 28 µg/ml and cisp (2, 4, 8 µm) were examined and photographed by binocular microscope. Viable cells (full arrow), morphological changes (dotted line), bars: 50 µm (magnification 100x). Effect of EFC and Cisp on Cells Death Induction on HeLa Cells According to the cytotoxic combination between EFC and cisp mentioned below, cell death detection was carried out by flow cytometry using the less of IC 50 concentration. Figure 3 shows the effect of EFC, cisp and combination of both caused cell death induction. EFC could not increase cell death through apoptosis signal. However, it could increase necrosis cell death which was induced by combination of EFC and cisp compared to cisp-single treatment. Cisp-single treatment and combination of cisp with EFC (24 h treatment) caused 82% and 72% cell death, respectively (Figure 3B). The apoptosis value of cisp-single treatment and its combination with EFC were 71.50% and 40.41%, while the necrosis values were 10.38% and 31.81%, respectively. EFC-single treatment caused less cell death induction with apoptosis value of 5.85% and necrosis value of 2.38%. These results suggest that EFC may affect HeLa cells sensitivity to cisp which is caused by apoptosis and necrosis cell death 55

6 (A) control 20 µg/ml EFC 8 µm Cisp 20 µg/ml EFC + 8 µm Cisp R4 R3 Sample ID: CONTROL HeLA 4 R3 Patient ID: Gated Events: Sample ID: ET OAC SECANG H Patient 4 R3 ID: Gated Events: Sample ID: CISPLATIN HeLA Patient 4 ID: Gated Events: R3 Sample ID: ET OAC SECANG+C Patient ID: Gated Events: R1 R2 Quad % Gated % Total UL UR LL LR R2 Quad % Gated % Total UL UR LL LR R2 Quad % Gated % Total UL UR LL LR R2 Quad % Gated % Total UL UR LL LR Annexin V-FITC (B) necrosis late apoptosis early apoptosis 2,38 1,39 4,46 EFC (µg/ml) Cisp (µm) Figure 3. Cells death induction was caused by Cisp and its combination with EFC. (A) HeLa cells was treated with EFC 20 μg/ml, Cisp 8 μm, and both of it for 24 h. Then, Cells death was calculated by Flow cytometry assay using annexin V-FITC/PI staining. R1 viable, R2 early apoptotic, R3 late apoptotic, R3 necrotic cells. (B) Data was analyzed using Microsoft exel 2007 describing cell death percentage. Effect of EFC and Cisp on Cell Cycle Modulation of HeLa Cells Cell Cycle analysis by flow cytometry was used to evaluate the cell cycle distribution by EFC, cisp and combination of both. Cisp has been reported to induce cell cycle arrest in G2/M phase in some cancer cells (Fehrmann and Laimins, 2003) and this study confirm that EFC induced cell cycle arrest in S phase. Cell distribution analysis was performed to investigate the effect of EFC addition to cisp on cell cycle arrest of HeLa cells. Figure 4 shows HeLa cells treated with 28 µg/ml EFC, 8 µm cisp, and a combination of the two agents for 24 h. HeLa cells treated with EFC and cisp showed cells accumulation in S phase and G2/M phase, respectively. While combination of both agents caused redistribution of the cell cycle in G1 and S phase compared to cisp-treated cells. In addition, accumulation of cells in G2/M phase decreased compared to cisptreated cells. These results above suggested that EFC enhances cytotoxicity induced by cisp on HeLa cells through cell cycle arrest in G1 and S phase. The cells progression in G1 and S phase might be caused by some proteins that play a role in cell cycle checkpoints. 56

7 Number control G % S 11.34% G2/M 6.79% Channels (FL2-A-FL2-Area) Number μg/ml EFC Apoptosis Dip G1 Dip G2 Dip S Channels (FL2-A-FL2-Area) File analy zed: ETOAC SECANG SEL HELA.003 Date analy zed: 28-Feb-2013 Model: 1nn0A_DSF Analy sis ty pe: Manual analy sis G1 1.88% S 97.89% G2/M 0.24% Diploid: % Dip G1: 1.88 % at Dip G2: 0.24 % at Dip S: % G2/G1: 4.97 %CV: Total S-Phase: % Total B.A.D.: 0.00 % no debris no aggs Apoptosis: % Mean: Debris: % Aggregates: 0.00 % Modeled ev ents: All cy cle ev ents: 9057 Cy cle ev ents per channel: 125 RCS: Figure 4. Cisp in combination with EFC caused cell cycle modulation on Hela cells. Cells were treated with 28 μg/ml EFC, 8μM cisp and combination of both for 24 h. Cell cycle distribution was calculated by Flow cytometry then analyzed using ModFit LT 3.0. Number DNA Content 8 μm cisp G % S 0.00 % G2/M 73.41% Channels (FL2-A-FL2-Area) Number μg/ml EFC + 8 μm cisp Channels (FL2-A-FL2-Area) G % S 22.97% G2/M 40.15% Expression of p53 was caused by EFC and Cisp on HeLa Cells To confirm the mechanism of EFC and R1 its combination with cisp in cell cycle arrest, therefore molecular target protein p53 was investigated. HeLa cells performed degradation and function loss FL2-A-FL2-Area of p53 as tumor suppressor protein which then cause uncontrolled proliferative ModFitLT V3.0(Win32) cells (Fehrmann and Laimins, 2003; Minaguchi et al., 1998). As shown in Figure 5, immunocytochemistry FL2-W-FL2-Width evaluation indicated that p53 level increased significantly (P < 0.05) (Figure 5A) confirmed by an intensive brown color in nucleus after being treated with 28 µg/ml EFC-single treatment and in combination with 8 µm cisp compared to cisp-single treatment and non-treated cells. The results suggest that EFC enhances cisp cytotoxicity on HeLa cells through p53 stabilization that perhaps play a role in cell cycle checkpoints. (A) (B) control 28 μg/ml EFC 8 μm cisp 28 μg/ml EFC + 8 μm cisp 65% p53 active (%) level of p53 24% 4% EFC 28µg/ml Cisp 8µM Figure 5. Effect of EFC and Cisp on p53 expression of HeLa cells. (A) HeLa cells were treated for 15 h with 28 µg/ml EFC, 8 µm cisp, and in combination of both. Data was examined and photographed by binocular microscope. Bars: 5 µm (magnification 400x). (B) Quantitative results showed analysis of p53 level in nucleus considered significantly different between combination treatment and single treatment (P < 0.05). 57

8 DISCUSSION Ethyl acetate fraction of C. sappan (EFC) performed more potent cytotoxic effect on HeLa cells compared to ethanolic extract (320 µg/ml, data not shown), showing a concentration dependent manner, with IC 50 of 65 ± 1.5 µg/ml. Combination treatment of EFC and cisp at low concentration performed significant inhibition of cell viability compared to cisp-single treatment. A relatively low concentration was used to minimize the additional side effects and cellular resistance of cisp. Decreasing cells viability might be due to either cell death or cell cycle arrest. This study shows that combination of EFC and cisp treatment on HeLa cells induced more necrotic rate up to 31.81% compared to cisp-single treatment. Afterward, EFC-single treatment caused less apoptosis compared to cisp. Hence, cisp is possibly more responsible in inducing apoptosis than EFC and its combination with EFC. The combination between EFC and cisp may affect on cell cycle modulation that can cause a decrease in cell viability on HeLa cells. Cisp has been reported as anti-cancer agent by inducing G2/M phase arrest and apoptosis in some type of cancer cells (Jamieson and Lippard, 1999; Fehrmann and Laimins, 2003). Our results suggest that EFC enhanced HeLa cells sensitivity to cisp through cell cycle redistribution, causing arrest in G1 and S phase. Cell cycle arrest is related to the efficacy of cytotoxicity of chemotherapeutic agent being used. Previous studies reported chemo-sensitization of cancer cells by many chemotherapeutic drugs perhaps due to cell cycle modulation (Lawrence et al., 2003). Therefore, the delay of cell cycle distribution in G1 and S phase by EFC may be important in the sensitization of HeLa cells to cisp which causes a delay in G2/M phase. The experiments was continued to investigate the protein target on cell cycle by detecting the presence of p53. HeLa cells are characterized with p53 degradation due to the binding of HPV E6 protein complexes to cellular factor E6AP which leads to ubiquitin-mediated degradation by proteasome (Buitrago-pérez et al., 2009). EFC increased p53 expression in nucleus and its combination with cisp significantly increased p53 expression compared to EFC and cisp-single treatment (Figure 5). The p53 protein activation may indirectly affect cellular response through some protein regulation causing cell cycle arrest to lead DNA repair or cell death activation (Jamieson and Lippard, 1999; Ricci and Zong, 2006; Schmitt et al., 2002). These results suggested that the mechanism of EFC in sensitizing HeLa cells to cisp was possibly via p53-dependent. Brazilein as the main compound of C. sappan probably could inhibit HPV E6 interaction with E6-binding protein E6AP. Therefore, brazilein may be amendable as HPV inhibitor agent and as competitor of vaccine to prevent the development of cervical cancer. However, this hypothesis still needs further investigation by in silico study. CONCLUSION In conclusion, EFC exhibits potential ability to enhance sensitivity of cervical cancer HeLa cells to cisp in vitro through cell cycle arrest with p53 stabilization. Further molecular target detection to investigate its cellular pathway needs to be conducted. ACKNOWLEDGEMENTS This study was partly supported by Grant from Hibah Penelitian Kerjasama Institusi, Universitas Gadjah Mada, Yogyakarta, Indonesia. We would like to especially thank to Prof. dr. Sofia Mubarika H., M. Med.Sc., Ph.D. (Faculty of Medicine) and Dr. Rarastoeti Pratiwi, M.Sc., Ph.D. (Faculty of Biology, Universitas Gadjah Mada) for the suggestion and correction. 58

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